Field of the Invention
This invention relates to immunoassaying. Immunoassayings are proving a immense value in medicine and biology for the assaying of the constituents of biological fluids, because of the sensitivity and specificity of such assaying. In immunoassaying procedures, for a given target compound, a synthetic antigen is generally first prepared. Heretofore, this has usually been accomplished by coupling the target compound, through a coupling group to a carrier which confers antigenicity to the entire compound. The compound coupled to the carrier is usually known as a hapten and, when coupled, it exhibits antigenic determinacy by causing the antibodies produced to be specific to it. Thus, the antibodies produced have a distinct and unique character, such that they will bind with only a specific compound or class of compounds. The objective in devising the synthetic antigen-hapten conjugate is to provide a compound which will generate antibodies that are specific to the target compound.
Antibodies are prepared by injecting the synthetic hapten-antigen conjugate into competent vertebrate animals and recovering blood serum from the animals after they have had time to generate antibodies. Typical and preferred animals are mammals, e.g., rabbits and goats.
The principal problem is usually synthesizing antigens that are sufficiently specific. Biological fluids such as blood and urine frequently contain very closely related compounds and it is a common for antibodies to be unable to distinguish the target compound from close relatives, or sometimes even distant ones. The antibody is then considered to be a poor one and is said to have low specificity and high cross-reactivity.
The assay itself is commonly a competitive binding assay. In such an assay, the target compound, which is not necessarily extracted, is allowed to compete with known quantities of a radiation labeled standard to bind with a known quantity of a specific antibody. From measurement of the proportion of the labeling in the standard-antibody complex that results, the amount of target compound present can be calculated. Radioactive labeling is particularly convenient. Fluoroescent perturbation can be used. Normally it will be necessary to remove any unreacted labeled standard, before making the determination on the antibody complex.